bims-cagime Biomed News
on Cancer, aging and metabolism
Issue of 2021‒01‒31
sixty-nine papers selected by
Kıvanç Görgülü
Technical University of Munich

  1. Elife. 2021 Jan 29. pii: e62852. [Epub ahead of print]10
    Yousefzadeh M, Henpita C, Vyas R, Soto-Palma C, Robbins P, Niedernhofer L.
      Aging is a complex process that results in loss of the ability to reattain homeostasis following stress, leading, thereby, to increased risk of morbidity and mortality. Many factors contribute to aging, such as the time-dependent accumulation of macromolecular damage, including DNA damage. The integrity of the nuclear genome is essential for cellular, tissue, and organismal health. DNA damage is a constant threat because nucleic acids are chemically unstable under physiological conditions and vulnerable to attack by endogenous and environmental factors. To combat this, all organisms possess highly conserved mechanisms to detect and repair DNA damage. Persistent DNA damage (genotoxic stress) triggers signaling cascades that drive cells into apoptosis or senescence to avoid replicating a damaged genome. The drawback is that these cancer avoidance mechanisms promote aging. Here, we review evidence that DNA damage plays a causal role in aging. We also provide evidence that genotoxic stress is linked to other cellular processes implicated as drivers of aging, including mitochondrial and metabolic dysfunction, altered proteostasis and inflammation. These links between damage to the genetic code and other pillars of aging support the notion that DNA damage could be the root of aging.
    Keywords:  Aging; DNA damage; DNA repair; genetics; genome instability; genomics; progeria
  2. Clin Cancer Res. 2021 Jan 25. pii: clincanres.3715.2020. [Epub ahead of print]
    Steele NG, Biffi G, Kemp SB, Zhang Y, Drouillard D, Syu LJ, Hao Y, Oni TE, Brosnan E, Elyada E, Doshi A, Hansma C, Espinoza C, Abbas A, The S, Irizarry-Negron VM, Halbrook CJ, Franks N, Hoffman M, Brown KL, Carpenter ES, Nwosu ZC, Johnson C, Lima F, Anderson MA, Park Y, Crawford HC, Lyssiotis CA, Frankel TL, Rao A, Bednar F, Dlugosz AA, Preall J, Tuveson DA, Allen B, Pasca di Magliano M.
      PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease characterized by an extensive fibroinflammatory stroma, which includes abundant cancer-associated fibroblast (CAF) populations. PDAC CAFs are heterogeneous, but the nature of this heterogeneity is incompletely understood. The Hedgehog (HH) pathway functions in PDAC in a paracrine manner, with ligands secreted by cancer cells signaling to stromal cells in the microenvironment. Previous reports investigating the role of HH signaling in PDAC have been contradictory, with HH signaling alternately proposed to promote or restrict tumor growth. In light of the newly discovered CAF heterogeneity, we investigated how HH pathway inhibition reprograms the PDAC microenvironment.EXPERIMENTAL DESIGN: We used a combination of pharmacologic inhibition, gain- and loss- of-function genetic experiments, CyTOF, and single cell RNA-sequencing to study the roles of HH signaling in PDAC.
    RESULTS: We find that HH signaling is uniquely activated in fibroblasts and differentially elevated in myofibroblastic CAFs (myCAFs) compared to inflammatory CAFs (iCAFs). SHH overexpression promotes tumor growth, while HH pathway inhibition with the Smoothened antagonist LDE225 impairs tumor growth. Further, HH pathway inhibition reduces myCAF numbers and increases iCAF numbers, which correlates with a decrease in cytotoxic T cells and an expansion in regulatory T cells, consistent with increased immune suppression.
    CONCLUSIONS: HH pathway inhibition alters fibroblast composition and immune infiltration in the pancreatic cancer microenvironment.
  3. Cancer Cell. 2021 Jan 09. pii: S1535-6108(20)30671-1. [Epub ahead of print]
    Lin H, Kryczek I, Li S, Green MD, Ali A, Hamasha R, Wei S, Vatan L, Szeliga W, Grove S, Li X, Li J, Wang W, Yan Y, Choi JE, Li G, Bian Y, Xu Y, Zhou J, Yu J, Xia H, Wang W, Alva A, Chinnaiyan AM, Cieslik M, Zou W.
      Immunotherapy induces durable clinical responses in a fraction of patients with cancer. However, therapeutic resistance poses a major challenge to current immunotherapies. Here, we identify that expression of tumor stanniocalcin 1 (STC1) correlates with immunotherapy efficacy and is negatively associated with patient survival across diverse cancer types. Gain- and loss-of-function experiments demonstrate that tumor STC1 supports tumor progression and enables tumor resistance to checkpoint blockade in murine tumor models. Mechanistically, tumor STC1 interacts with calreticulin (CRT), an "eat-me" signal, and minimizes CRT membrane exposure, thereby abrogating membrane CRT-directed phagocytosis by antigen-presenting cells (APCs), including macrophages and dendritic cells. Consequently, this impairs APC capacity of antigen presentation and T cell activation. Thus, tumor STC1 inhibits APC phagocytosis and contributes to tumor immune evasion and immunotherapy resistance. We suggest that STC1 is a previously unappreciated phagocytosis checkpoint and targeting STC1 and its interaction with CRT may sensitize to cancer immunotherapy.
    Keywords:  PD-1; T cell immunity; calreticulin; checkpoint; dendritic cell; eat-me signal; macrophages; phagocytosis; stanniocalcin 1; tumor
  4. Nat Immunol. 2021 Jan 28.
    Rai P, Janardhan KS, Meacham J, Madenspacher JH, Lin WC, Karmaus PWF, Martinez J, Li QZ, Yan M, Zeng J, Grinstaff MW, Shirihai OS, Taylor GA, Fessler MB.
      Mitochondrial abnormalities have been noted in lupus, but the causes and consequences remain obscure. Autophagy-related genes ATG5, ATG7 and IRGM have been previously implicated in autoimmune disease. We reasoned that failure to clear defective mitochondria via mitophagy might be a foundational driver in autoimmunity by licensing mitochondrial DNA-dependent induction of type I interferon. Here, we show that mice lacking the GTPase IRGM1 (IRGM homolog) exhibited a type I interferonopathy with autoimmune features. Irgm1 deletion impaired the execution of mitophagy with cell-specific consequences. In fibroblasts, mitochondrial DNA soiling of the cytosol induced cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-dependent type I interferon, whereas in macrophages, lysosomal Toll-like receptor 7 was activated. In vivo, Irgm1-/- tissues exhibited mosaic dependency upon nucleic acid receptors. Whereas salivary and lacrimal gland autoimmune pathology was abolished and lung pathology was attenuated by cGAS and STING deletion, pancreatic pathology remained unchanged. These findings reveal fundamental connections between mitochondrial quality control and tissue-selective autoimmune disease.
  5. Anal Chem. 2021 Jan 27.
    Lozano-Torres B, Blandez JF, Galiana I, Lopez-Dominguez JA, Rovira M, Paez-Ribes M, González-Gualda E, Muñoz-Espín D, Serrano M, Sancenón F, Martínez-Máñez R.
      Cellular senescence is a state of stable cell cycle arrest that can negatively affect the regenerative capacities of tissues and can contribute to inflammation and the progression of various aging-related diseases. Advances in the in vivo detection of cellular senescence are still crucial to monitor the action of senolytic drugs and to assess the early onset or accumulation of senescent cells. Here, we describe a naphthalimide-styrene-based probe (HeckGal) for the detection of cellular senescence both in vitro and in vivo. HeckGal is hydrolyzed by the increased lysosomal β-galactosidase activity of senescent cells, resulting in fluorescence emission. The probe was validated in vitro using normal human fibroblasts and various cancer cell lines undergoing senescence induced by different stress stimuli. Remarkably, HeckGal was also validated in vivo in an orthotopic breast cancer mouse model treated with senescence-inducing chemotherapy and in a renal fibrosis mouse model. In all cases, HeckGal allowed the unambiguous detection of senescence in vitro as well as in tissues and tumors in vivo. This work is expected to provide a potential technology for senescence detection in aged or damaged tissues.
  6. Cell. 2021 Jan 20. pii: S0092-8674(21)00002-7. [Epub ahead of print]
    Litchfield K, Reading JL, Puttick C, Thakkar K, Abbosh C, Bentham R, Watkins TBK, Rosenthal R, Biswas D, Rowan A, Lim E, Al Bakir M, Turati V, Guerra-Assunção JA, Conde L, Furness AJS, Saini SK, Hadrup SR, Herrero J, Lee SH, Van Loo P, Enver T, Larkin J, Hellmann MD, Turajlic S, Quezada SA, McGranahan N, Swanton C.
      Checkpoint inhibitors (CPIs) augment adaptive immunity. Systematic pan-tumor analyses may reveal the relative importance of tumor-cell-intrinsic and microenvironmental features underpinning CPI sensitization. Here, we collated whole-exome and transcriptomic data for >1,000 CPI-treated patients across seven tumor types, utilizing standardized bioinformatics workflows and clinical outcome criteria to validate multivariable predictors of CPI sensitization. Clonal tumor mutation burden (TMB) was the strongest predictor of CPI response, followed by total TMB and CXCL9 expression. Subclonal TMB, somatic copy alteration burden, and histocompatibility leukocyte antigen (HLA) evolutionary divergence failed to attain pan-cancer significance. Dinucleotide variants were identified as a source of immunogenic epitopes associated with radical amino acid substitutions and enhanced peptide hydrophobicity/immunogenicity. Copy-number analysis revealed two additional determinants of CPI outcome supported by prior functional evidence: 9q34 (TRAF2) loss associated with response and CCND1 amplification associated with resistance. Finally, single-cell RNA sequencing (RNA-seq) of clonal neoantigen-reactive CD8 tumor-infiltrating lymphocytes (TILs), combined with bulk RNA-seq analysis of CPI-responding tumors, identified CCR5 and CXCL13 as T-cell-intrinsic markers of CPI sensitivity.
    Keywords:  CXCL9; biomarkers; checkpoint inhibitors; clonal TMB; immunogenicity; immunotherapy; meta-analysis; mutation; neoantigen
  7. Autophagy. 2021 Jan 26. 1-3
    Lee C, Overholtzer M.
      Lysosomes play an essential role in quality control mechanisms by functioning as the primary digestive system in mammalian cells. However, the quality control mechanisms governing healthy lysosomes are not fully understood. Using a method to study lysosome membrane turnover, we discovered that LC3-lipidation on the lysosome limiting membrane is involved in invagination and formation of intralumenal vesicles, an activity known as microautophagy. This activity occurs in response to metabolic stress, in the form of glucose starvation, or osmotic stress induced by treatment with lysosomotropic compounds. Cells rendered deficient in the ability to lipidate LC3 through knockout of ATG5 show reduced ability to regulate lysosome size and degradative function in response to stress. These findings demonstrate that cells can adapt to changing metabolic conditions by turning over selective portions of the lysosomal membrane, using a mechanism that involves lysosome-targeted LC3 lipidation and the induction of selective microautophagy.
    Keywords:  ATG5; LC3; autophagy; lysophagy; microautophagy
  8. Trends Cancer. 2021 Jan 25. pii: S2405-8033(20)30341-1. [Epub ahead of print]
    Ferguson LP, Diaz E, Reya T.
      Despite gains in knowledge of the intrinsic signals governing cancer progression, effective clinical management of cancer remains a challenge. Drug resistance and relapse, pose the greatest barriers to cancer care, and are often driven by the co-option of stem cell programs by subpopulations of aggressive cancer cells. Here, we focus on the role of the microenvironment in the acquisition and/or maintenance of stem cell states in cancer in the context of resistance and metastasis. We further discuss the role of cancer stem cells in immune evasion through the course of metastasis, dormancy, and relapse. Understanding the niche in which cancer stem cells live and the signals that sustain them may lead to new strategies that target them by disrupting microenvironmental support.
    Keywords:  Cancer; Metastasis; Stem cell; Therapy resistance; Tumor microenvironment
  9. Trends Mol Med. 2021 Jan 21. pii: S1471-4914(20)30329-4. [Epub ahead of print]
    Dan J, Memczak S, Izpisua Belmonte JC.
      Genome editing holds great promise for treating a range of human genetic diseases. While emerging clustered regularly interspaced short-palindromic repeats (CRISPR) technologies allow editing of the nuclear genome, it is still not possible to precisely manipulate mitochondrial DNA (mtDNA). Here, we summarize past developments and recent advances in nuclear and mitochondrial genome editing.
    Keywords:  gene therapy; genome editing; mitochondria; translational medicine
  10. Cell. 2021 Jan 18. pii: S0092-8674(20)31694-9. [Epub ahead of print]
    Prentzell MT, Rehbein U, Cadena Sandoval M, De Meulemeester AS, Baumeister R, Brohée L, Berdel B, Bockwoldt M, Carroll B, Chowdhury SR, von Deimling A, Demetriades C, Figlia G, , de Araujo MEG, Heberle AM, Heiland I, Holzwarth B, Huber LA, Jaworski J, Kedra M, Kern K, Kopach A, Korolchuk VI, van 't Land-Kuper I, Macias M, Nellist M, Palm W, Pusch S, Ramos Pittol JM, Reil M, Reintjes A, Reuter F, Sampson JR, Scheldeman C, Siekierska A, Stefan E, Teleman AA, Thomas LE, Torres-Quesada O, Trump S, West HD, de Witte P, Woltering S, Yordanov TE, Zmorzynska J, Opitz CA, Thedieck K.
      Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.
    Keywords:  G3BP1; G3BP2; TSC complex; cancer; lysosome; mTORC1; metabolism; neuronal function; stress granule
  11. JCI Insight. 2021 Jan 25. pii: 136176. [Epub ahead of print]6(2):
    Aftabizadeh M, Li YJ, Zhao Q, Zhang C, Ambaye N, Song J, Nagao T, Lahtz C, Fakih M, Ann DK, Yu H, Herrmann A.
      To date, there are no inhibitors that directly and specifically target activated STAT3 and c-Myc in the clinic. Although peptide-based inhibitors can selectively block activated targets, their clinical usage is limited because of low cell penetration and/or serum stability. Here, we generated cell-penetrating acetylated (acet.) STAT3, c-Myc, and Gp130 targeting peptides by attaching phosphorothioated (PS) polymer backbone to peptides. The cell-penetrating peptides efficiently penetrated cells and inhibited activation of the intended targets and their downstream genes. Locally or systemically treating tumor-bearing mice with PS-acet.-STAT3 peptide at low concentrations effectively blocked STAT3 in vivo, resulting in significant antitumor effects in 2 human xenograft models. Moreover, PS-acet.-STAT3 peptide penetrated and activated splenic CD8+ T cells in vitro. Treating immune-competent mice bearing mouse melanoma with PS-acet.-STAT3 peptide inhibited STAT3 in tumor-infiltrating T cells, downregulating tumor-infiltrating CD4+ T regulatory cells while activating CD8+ T effector cells. Similarly, systemic injections of the cell-penetrating c-Myc and Gp130 peptides prevented pancreatic tumor growth and induced antitumor immune responses. Taken together, we have developed therapeutic peptides that effectively and specifically block challenging cancer targets, resulting in antitumor effects through both direct tumor cell killing and indirectly through antitumor immune responses.
    Keywords:  Oncology; Peptides; Therapeutics
  12. J Biol Chem. 2021 Jan 23. pii: S0021-9258(21)00106-X. [Epub ahead of print] 100335
    Eiyama A, Aaltonen MJ, Nolte H, Tatsuta T, Langer T.
      Lipid transfer proteins of the Ups1/PRELID1 family facilitate the transport of phospholipids across the intermembrane space of mitochondria in a lipid-specific manner. Heterodimeric complexes of yeast Ups1/Mdm35 or human PRELID1/TRIAP1 shuttle phosphatidic acid (PA) mainly synthesized in the endoplasmic reticulum (ER) to the inner membrane, where it is converted to cardiolipin (CL), the signature phospholipid of mitochondria. Loss of Ups1/PRELID1 proteins impairs the accumulation of CL and broadly affects mitochondrial structure and function. Unexpectedly and unlike yeast cells lacking the cardiolipin synthase Crd1, Ups1 deficient yeast cells exhibit glycolytic growth defects, pointing to functions of Ups1-mediated PA transfer beyond CL synthesis. Here, we show that the disturbed intramitochondrial transport of PA in ups1Δ cells leads to altered unfolded protein response (UPR) and mTORC1 signaling, independent of disturbances in CL synthesis. The impaired flux of PA into mitochondria is associated with the increased synthesis of phosphatidylcholine (PC) and a reduced phosphatidylethanolamine (PE)/PC ratio in the ER of ups1Δ cells which suppresses the UPR. Moreover, we observed inhibition of TORC1 signaling in these cells. Activation of either UPR by ER protein stress or of TORC1 signaling by disruption of its negative regulator, the SEACIT complex, increased cytosolic protein synthesis and restored glycolytic growth of ups1Δ cells. These results demonstrate that PA influx into mitochondria is required to preserve ER membrane homeostasis and that its disturbance is associated with impaired glycolytic growth and cellular stress signaling.
    Keywords:  Mitochondria; PRELID1; TORC1; Ups1; endoplasmic reticulum (ER); lipid transfer; phospholipid; unfolded protein response (UPR); yeast
  13. J Biol Chem. 2020 Feb 07. pii: S0021-9258(17)49861-9. [Epub ahead of print]295(6): 1623-1636
    Kuwana T, King LE, Cosentino K, Suess J, Garcia-Saez AJ, Gilmore AP, Newmeyer DD.
      Permeabilization of the mitochondrial outer membrane is a key step in the intrinsic apoptosis pathway, triggered by the release of mitochondrial intermembrane space proteins into the cytoplasm. The BCL-2-associated X apoptosis regulator (BAX) protein critically contributes to this process by forming pores in the mitochondrial outer membrane. However, the relative roles of the mitochondrial residence of BAX and its oligomerization in promoting membrane permeabilization are unclear. To this end, using both cell-free and cellular experimental systems, including membrane permeabilization, size-exclusion chromatography-based oligomer, and retrotranslocation assays, along with confocal microscopy analysis, here we studied two BAX C-terminal variants, T182I and G179P. Neither variant formed large oligomers when activated in liposomes. Nevertheless, the G179P variant could permeabilize liposome membranes, suggesting that large BAX oligomers are not essential for the permeabilization. However, when G179P was transduced into BAX/BCL2 agonist killer (BAK) double-knockout mouse embryonic fibroblasts, its location was solely cytoplasmic, and it then failed to mediate cell death. In contrast, T182I was inefficient in both liposome insertion and permeabilization. Yet, when transduced into cells, BAXT182I resided predominantly on mitochondria, because of its slow retrotranslocation and mediated apoptosis as efficiently as WT BAX. We conclude that BAX's mitochondrial residence in vivo, regulated by both targeting and retrotranslocation, is more significant for its pro-apoptotic activity than its ability to insert and to form higher-order oligomers in model membranes. We propose that this finding should be taken into account when developing drugs that modulate BAX activity.
    Keywords:  Bax; anticancer drug; apoptosis; liposome; mitochondrial apoptosis; mitochondrial localization; mitochondrial outer membrane permeabilization (MOMP); molecular cell biology; protein oligomers; translocation
  14. Elife. 2021 Jan 29. pii: e63728. [Epub ahead of print]10
    Miller KN, Dasgupta N, Liu T, Adams PD, Vizioli MG.
      Senescent cells, damaged cells that permanently exit the cell cycle, play important roles in development, tissue homeostasis, and tumorigenesis. Although many of these roles are beneficial in acute responses to stress and damage, the persistent accumulation of senescent cells is associated with many chronic diseases through their proinflammatory senescence-associated secretory phenotype (SASP). SASP expression is linked to DNA damage; however, the mechanisms that control the SASP are incompletely understood. More recently, it has been shown that senescent cells shed fragments of nuclear chromatin into the cytoplasm, so called cytoplasmic chromatin fragments (CCF). Here, we provide an overview of the current evidence linking DNA damage to the SASP through the formation of CCF. We describe mechanisms of CCF generation and their functional role in senescent cells, with emphasis on therapeutic potential.
    Keywords:  aging; cell biology; chromosomes; cytoplasmic chromatin fragments; epigenetics; gene expression; mitochondria; senescence
  15. Metabolites. 2021 Jan 22. pii: 64. [Epub ahead of print]11(2):
    Paley S, Billington R, Herson J, Krummenacker M, Karp PD.
      Metabolomics, synthetic biology, and microbiome research demand information about organism-scale metabolic networks. The convergence of genome sequencing and computational inference of metabolic networks has enabled great progress toward satisfying that demand by generating metabolic reconstructions from the genomes of thousands of sequenced organisms. Visualization of whole metabolic networks is critical for aiding researchers in understanding, analyzing, and exploiting those reconstructions. We have developed bioinformatics software tools that automatically generate a full metabolic-network diagram for an organism, and that enable searching and analyses of the network. The software generates metabolic-network diagrams for unicellular organisms, for multi-cellular organisms, and for pan-genomes and organism communities. Search tools enable users to find genes, metabolites, enzymes, reactions, and pathways within a diagram. The diagrams are zoomable to enable researchers to study local neighborhoods in detail and to see the big picture. The diagrams also serve as tools for comparison of metabolic networks and for interpreting high-throughput datasets, including transcriptomics, metabolomics, and reaction fluxes computed by metabolic models. These data can be overlaid on the metabolic charts to produce animated zoomable displays of metabolic flux and metabolite abundance. The website contains whole-network diagrams for more than 18,000 sequenced organisms. The ready availability of organism-specific metabolic network diagrams and associated tools for almost any sequenced organism are useful for researchers working to better understand the metabolism of their organism and to interpret high-throughput datasets in a metabolic context.
    Keywords:  biochemical pathways charts; metabolic charts; metabolic diagrams; metabolic maps; metabolic network diagrams; metabolomics; transcriptomics
  16. Elife. 2021 Jan 25. pii: e61082. [Epub ahead of print]10
    Afsari B, Kuo A, Zhang Y, Li L, Lahouel K, Danilova L, Favorov A, Rosenquist TA, Grollman AP, Kinzler KW, Cope L, Vogelstein B, Tomasetti C.
      Determining the etiologic basis of the mutations that are responsible for cancer is one of the fundamental challenges in modern cancer research. Different mutational processes induce different types of DNA mutations, providing 'mutational signatures' that have led to key insights into cancer etiology. The most widely used signatures for assessing genomic data are based on unsupervised patterns that are then retrospectively correlated with certain features of cancer. We show here that supervised machine-learning techniques can identify signatures, called SuperSigs, that are more predictive than those currently available. Surprisingly, we found that aging yields different SuperSigs in different tissues, and the same is true for environmental exposures. We were able to discover SuperSigs associated with obesity, the most important lifestyle factor contributing to cancer in Western populations.
    Keywords:  cancer biology; human
  17. J Gastrointest Surg. 2021 Jan 26.
    Michelakos T, Sekigami Y, Kontos F, Fernández-Del Castillo C, Qadan M, Deshpande V, Ting DT, Clark JW, Weekes CD, Parikh A, Ryan DP, Wo JY, Hong TS, Allen JN, Catalano O, Warshaw AL, Lillemoe KD, Ferrone CR.
      BACKGROUND: Dynamic survival data based on time already survived are lacking for resected borderline resectable/locally advanced (BR/LA) pancreatic ductal adenocarcinoma (PDAC) patients who received total neoadjuvant therapy (TNT) with FOLFIRINOX followed by chemoradiation. Conditional survival, i.e., the probability of surviving an additional length of time after having already survived an amount of time, offers such information. We aimed to determine actuarial and conditional overall (OS, COS) and disease-free survival (DFS, CDFS) among this cohort.METHODS: Clinicopathologic data were retrospectively collected for resected BR/LA PDAC patients who received TNT (2011-2019). COS and CDFS rates were calculated for patients being event (death/recurrence)-free at multiple intervals and by recurrence status.
    RESULTS: After a median follow-up of 32.1 months, the 183 patients had a median OS and DFS of 39.1 months and 16.8 months, respectively. COS and CDFS increased as a function of time already survived. The probability of surviving an additional 24 months if a patient survived 2 years post-operatively was 70%, whereas the 4-year actuarial OS was 47%. Similarly, the probability of surviving disease-free an additional 24 months after 2 years was 66%, while actuarial 48-month DFS was 27%. COS for disease-free patients increased further over time. For patients remaining disease-free 12 months post-operatively, BR vs. LA status at diagnosis, tumor ≤ 4 cm at diagnosis, and R0 resection were independent predictors of favorable additional OS and DFS.
    CONCLUSIONS: For resected TNT-treated BR/LA PDAC patients, the probability of surviving an additional length of time increases as a function of survival already accrued. Dynamic survival estimates may allow personalized follow-up and counseling.
    Keywords:  Conditional survival; FOLFIRINOX; Pancreatic ductal adenocarcinoma; Total neoadjuvant therapy
  18. Elife. 2021 Jan 28. pii: e60710. [Epub ahead of print]10
    Lehtimäki JI, Rajakylä EK, Tojkander S, Lappalainen P.
      Contractile actomyosin bundles, stress fibers, govern key cellular processes including migration, adhesion, and mechanosensing. Stress fibers are thus critical for developmental morphogenesis. The most prominent actomyosin bundles, ventral stress fibers, generated through coalescence of pre-existing stress fiber precursors. However, whether stress fibers can assemble through other mechanisms has remained elusive. We report that stress fibers can also form without requirement of pre-existing actomyosin bundles. These structures, which we named cortical stress fibers, are embedded in the cell cortex and assemble preferentially underneath the nucleus. In this process, non-muscle myosin II pulses orchestrate the reorganization of cortical actin meshwork into regular bundles, which promote reinforcement of nascent focal adhesions, and subsequent stabilization of the cortical stress fibers. These results identify a new mechanism by which stress fibers can be generated de novo from the actin cortex, and establish role for stochastic myosin pulses in the assembly of functional actomyosin bundles.
    Keywords:  cell biology; none
  19. Cancers (Basel). 2021 Jan 23. pii: 427. [Epub ahead of print]13(3):
    Wang S, Prizment A, Thyagarajan B, Blaes A.
      Rapid improvements in cancer survival led to the realization that many modalities used to treat or control cancer may cause accelerated aging in cancer survivors. Clinically, "accelerated aging" phenotypes in cancer survivors include secondary cancers, frailty, chronic organ dysfunction, and cognitive impairment, all of which can impact long-term health and quality of life in cancer survivors. The treatment-induced accelerated aging in cancer survivors could be explained by telomere attrition, cellular senescence, stem cell exhaustion, DNA damage, and epigenetic alterations. Several aging clocks and biomarkers of aging have been proposed to be potentially useful in estimating biological age, which can provide specific information about how old an individual is biologically independent of chronological age. Measuring biological age in cancer survivors may be important for two reasons. First, it can better predict the risk of cancer treatment-related comorbidities than chronological age. Second, biological age may provide additional value in evaluating the effects of treatments and personalizing cancer therapies to maximize efficacy of treatment. A deeper understanding of treatment-induced accelerated aging in individuals with cancer may lead to novel strategies that reduce the accelerated aging and improve the quality of life in cancer survivors.
    Keywords:  accelerated aging; cancer treatment; cellular senescence
  20. Cancer Immunol Res. 2021 Jan 28. pii: canimm.0272.2020. [Epub ahead of print]
    Ajina R, Malchiodi ZX, Fitzgerald AA, Zuo A, Wang S, Moussa M, Cooper CJ, Shen Y, Johnson QR, Parks JM, Smith JC, Catalfamo M, Fertig EJ, Jablonski SA, Weiner LM.
      Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States. Pancreatic tumors are minimally infiltrated by T cells and are largely refractory to immunotherapy. Accordingly, the role of T-cell immunity in pancreatic cancer has been somewhat overlooked. Here, we hypothesized that immune resistance in pancreatic cancer was induced in response to antitumor T-cell immune responses, and that understanding how pancreatic tumors respond to immune attack may facilitate the development of more effective therapeutic strategies. We now provide evidence that T cell-dependent host immune responses induce a PDAC-derived myeloid mimicry phenomenon and stimulate immune resistance. Three KPC mouse models of pancreatic cancer were employed: the mT3-2D (Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre) subcutaneous and orthotopic models, as well as the KP1 (p48-CRE/LSL-Kras/Trp53flox/flox) subcutaneous model. KPC cancer cells were grown in immunocompetent and immunodeficient C57BL/6 mice and analyzed to determine the impact of adaptive immunity on malignant epithelial cells, as well as on whole tumors. We found that induced T-cell antitumor immunity, via signal transducer and activator of transcription 1 (STAT1), stimulated malignant epithelial pancreatic cells to induce the expression of genes typically expressed by myeloid cells and altered intratumoral immunosuppressive myeloid cell profiles. Targeting the Janus Kinase (JAK)/STAT signaling pathway using the FDA approved drug ruxolitinib overcame these tumor-protective responses and improved anti-PD-1 therapeutic efficacy. These findings provide future directions for treatments that specifically disable this mechanism of resistance in PDAC.
  21. Elife. 2021 Jan 27. pii: e57838. [Epub ahead of print]10
    Hart SFM, Chen CC, Shou W.
      Cooperation, paying a cost to benefit others, is widespread. Cooperation can be promoted by pleiotropic 'win-win' mutations which directly benefit self ('self-serving') and partner ('partner-serving'). Previously, we showed that partner-serving should be defined as increased benefit supply rate per intake benefit (Hart & Pineda et al., 2019). Here, we report that win-win mutations can rapidly evolve even under conditions unfavorable for cooperation. Specifically, in a well-mixed environment we evolved engineered yeast cooperative communities where two strains exchanged costly metabolites lysine and hypoxanthine. Among cells that consumed lysine and released hypoxanthine, ecm21 mutations repeatedly arose. ecm21 is self-serving, improving self's growth rate in limiting lysine. ecm21 is also partner-serving, increasing hypoxanthine release rate per lysine consumption and the steady state growth rate of partner. ecm21 also arose in monocultures evolving in lysine-limited chemostats. Thus, even without any history of cooperation or pressure to maintain cooperation, pleiotropic win-win mutations may readily evolve.
    Keywords:  S. cerevisiae; ecology; evolutionary biology
  22. Redox Biol. 2021 Jan 21. pii: S2213-2317(21)00018-5. [Epub ahead of print] 101870
    Boese AC, Kang S.
      Cancer cells display abnormal metabolic activity as a result of activated oncogenes and loss of tumor suppressor genes. The Warburg Effect is a common metabolic feature of cancer that involves a preference for aerobic glycolysis over oxidative phosphorylation to generate ATP and building blocks for biosynthesis. However, emerging evidence indicates that mitochondrial metabolic pathways are also reprogrammed in cancer and play vital roles in bioenergetics, biosynthesis, and managing redox homeostasis. The mitochondria act a central hub for metabolic pathways that generate ATP and building blocks for lipid, nucleic acid and protein biosynthesis. However, mitochondrial respiration is also a leading source of reactive oxygen species that can damage cellular organelles and trigger cell death if levels become too high. In general, cancer cells are reported to have higher levels of reactive oxygen species than their non-cancerous cells of origin, and therefore must employ diverse metabolic strategies to prevent oxidative stress. However, mounting evidence indicates that the metabolic profiles between proliferative and disseminated cancer cells are not the same. In this review, we will examine mitochondrial metabolic pathways, such as glutaminolysis, that proliferative and disseminated cancer cells utilize to control their redox status.
    Keywords:  Cancer progression; Glutaminolysis; Mitochondria metabolism; Redox homeostasis
  23. Nat Biotechnol. 2021 Jan 28.
    Prensner JR, Enache OM, Luria V, Krug K, Clauser KR, Dempster JM, Karger A, Wang L, Stumbraite K, Wang VM, Botta G, Lyons NJ, Goodale A, Kalani Z, Fritchman B, Brown A, Alan D, Green T, Yang X, Jaffe JD, Roth JA, Piccioni F, Kirschner MW, Ji Z, Root DE, Golub TR.
      Although genomic analyses predict many noncanonical open reading frames (ORFs) in the human genome, it is unclear whether they encode biologically active proteins. Here we experimentally interrogated 553 candidates selected from noncanonical ORF datasets. Of these, 57 induced viability defects when knocked out in human cancer cell lines. Following ectopic expression, 257 showed evidence of protein expression and 401 induced gene expression changes. Clustered regularly interspaced short palindromic repeat (CRISPR) tiling and start codon mutagenesis indicated that their biological effects required translation as opposed to RNA-mediated effects. We found that one of these ORFs, G029442-renamed glycine-rich extracellular protein-1 (GREP1)-encodes a secreted protein highly expressed in breast cancer, and its knockout in 263 cancer cell lines showed preferential essentiality in breast cancer-derived lines. The secretome of GREP1-expressing cells has an increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth-inhibitory effect of GREP1 knockout. Our experiments suggest that noncanonical ORFs can express biologically active proteins that are potential therapeutic targets.
  24. Trends Cancer. 2021 Jan 23. pii: S2405-8033(21)00016-9. [Epub ahead of print]
    Kesavan R, Rion H, Hoxhaj G.
      The molecular elements that govern cellular transformation and tumorigenic competence remain poorly understood. Metabolic reprogramming has emerged as a hallmark of malignant transformation. Recently in Cell Metabolism, Zhang et al. showed that an increase of cellular antioxidant capacity and nucleotide availability is sufficient to induce oncogenic transformation and tumorigenesis.
  25. Trends Cell Biol. 2021 Jan 20. pii: S0962-8924(20)30267-1. [Epub ahead of print]
    Suresh S, Huard S, Dubois T.
      Coactivator-associated arginine methyltransferase 1 (CARM1), identified 20 years ago as a coregulator of transcription, is an enzyme that catalyzes arginine methylation of proteins. Beyond its well-established involvement in the regulation of transcription, the physiological functions of CARM1 are still poorly understood. However, recent studies have revealed novel roles of CARM1 in autophagy, metabolism, paraspeckles, and early development. In addition, CARM1 is emerging as an attractive therapeutic target and a drug response biomarker for certain types of cancer. Here, we provide a comprehensive overview of the structure of CARM1 and its post-translational modifications, its various functions, apart from transcriptional coactivation, and its involvement in cancer.
    Keywords:  CARM1; PRMT4; arginine-methylation; autophagy; cancer; development
  26. JCI Insight. 2021 Jan 26. pii: 145389. [Epub ahead of print]
    Wattenberg MM, Herrera VM, Giannone MA, Gladney WL, Carpenter EL, Beatty GL.
      Agonistic anti-CD40 monoclonal antibody (mAb) therapy in combination with chemotherapy (chemoimmunotherapy) shows promise for the treatment of pancreatic ductal adenocarcinoma (PDA). To gain insight into immunological mechanisms of response and resistance to chemoimmunotherapy, we analyzed blood samples from patients (n=22) with advanced PDA treated with an anti-CD40 mAb (CP-870,893) in combination with gemcitabine. We found a stereotyped cellular response to chemoimmunotherapy characterized by transient B cell, CD56+CD11c+HLA-DR+CD141+ cell and monocyte depletion and CD4+ T cell activation. However, these cellular pharmacodynamics did not associate with outcomes. In contrast, we identified an inflammatory network in the peripheral blood consisting of neutrophils, cytokines (IL-6 and IL-8) and acute phase reactants (CRP and SAA) that was associated with outcomes. Furthermore, monocytes from patients with elevated plasma IL-6 and IL-8 showed distinct transcriptional profiles, including upregulation of CCR2 and GAS6; genes associated with regulation of leukocyte chemotaxis and response to inflammation. Patients with systemic inflammation, defined by neutrophil-lymphocyte ratio (NLR) >3.1, had a shorter median OS (5.8 vs 12.3mo; p=0.0105) as compared to patients with NLR <3.1. Taken together, our findings identify systemic inflammation as a potential resistance mechanism to a CD40-based chemoimmunotherapy and suggest biomarkers for future studies.
    Keywords:  Cancer immunotherapy; Cellular immune response; Immunology; Oncology
  27. Sci Adv. 2021 Jan;pii: eabd6449. [Epub ahead of print]7(5):
    Khatua B, El-Kurdi B, Patel K, Rood C, Noel P, Crowell M, Yaron JR, Kostenko S, Guerra A, Faigel DO, Lowe M, Singh VP.
      Obesity sometimes seems protective in disease. This obesity paradox is predominantly described in reports from the Western Hemisphere during acute illnesses. Since adipose triglyceride composition corresponds to long-term dietary patterns, we performed a meta-analysis modeling the effect of obesity on severity of acute pancreatitis, in the context of dietary patterns of the countries from which the studies originated. Increased severity was noted in leaner populations with a higher proportion of unsaturated fat intake. In mice, greater hydrolysis of unsaturated visceral triglyceride caused worse organ failure during pancreatitis, even when the mice were leaner than those having saturated triglyceride. Saturation interfered with triglyceride's interaction and lipolysis by pancreatic triglyceride lipase, which mediates organ failure. Unsaturation increased fatty acid monomers in vivo and aqueous media, resulting in greater lipotoxic cellular responses and organ failure. Therefore, visceral triglyceride saturation reduces the ensuing lipotoxicity despite higher adiposity, thus explaining the obesity paradox.
  28. Nat Chem Biol. 2021 Jan 25.
    Liu Y, Jurczak MJ, Lear TB, Lin B, Larsen MB, Kennerdell JR, Chen Y, Huckestein BR, Nguyen MK, Tuncer F, Jiang Y, Monga SP, O'Donnell CP, Finkel T, Chen BB, Mallampalli RK.
      The adenosine monophosphate (AMP)-activated protein kinase (Ampk) is a central regulator of metabolic pathways, and increasing Ampk activity has been considered to be an attractive therapeutic target. Here, we have identified an orphan ubiquitin E3 ligase subunit protein, Fbxo48, that targets the active, phosphorylated Ampkα (pAmpkα) for polyubiquitylation and proteasomal degradation. We have generated a novel Fbxo48 inhibitory compound, BC1618, whose potency in stimulating Ampk-dependent signaling greatly exceeds 5-aminoimidazole-4-carboxamide-1-β-ribofuranoside (AICAR) or metformin. This compound increases the biological activity of Ampk not by stimulating the activation of Ampk, but rather by preventing activated pAmpkα from Fbxo48-mediated degradation. We demonstrate that, consistent with augmenting Ampk activity, BC1618 promotes mitochondrial fission, facilitates autophagy and improves hepatic insulin sensitivity in high-fat-diet-induced obese mice. Hence, we provide a unique bioactive compound that inhibits pAmpkα disposal. Together, these results define a new pathway regulating Ampk biological activity and demonstrate the potential utility of modulating this pathway for therapeutic benefit.
  29. Cancer Lett. 2021 Jan 21. pii: S0304-3835(21)00031-8. [Epub ahead of print]
    Sagar S, Leiphrakpam PD, Thomas D, McAndrews KL, Caffrey TC, Swanson BJ, Clausen H, Wandall HH, Hollingsworth MA, Radhakrishnan P.
      Pancreatic ductal adenocarcinoma (PDAC) is highly lethal. MUC4 (mucin4) is a heavily glycosylated protein aberrantly expressed in PDAC and promotes tumorigenesis via an unknown mechanism. To assess this, we genetically knocked out (KO) MUC4 in PDAC cells that did not express and did express truncated O-glycans (Tn/STn) using CRISPR/Cas9 technology. We found that MUC4 knockout cells possess less tumorigenicity in vitro and in vivo, which was further reduced in PDAC cells that express aberrant overexpression of truncated O-glycans. Also, MUC4KO cells showed a further reduction of epidermal growth factor receptors (ErbB) and their downstream signaling pathways in truncated O-glycan expressing PDAC cells. Tn-MUC4 specific 3B11 antibody inhibited MUC4-induced ErbB receptor and its downstream signaling cascades. MUC4 knockout differentially regulates apoptosis and cell cycle arrest in branched and truncated O-glycan expressing PDAC cells. Additionally, MUC4KO cells were found to be more sensitive to gemcitabine treatment. They possessed the upregulated expression of hENT1 and hCNT3 compared to parental cells, which were further affected in cells with aberrant O-glycosylation. Taken together, our results indicate that MUC4 enhances the malignant properties and gemcitabine resistance in PDAC tumors that aberrantly overexpress truncated O-glycans via altering ErbB/AKT signaling cascades and expression of nucleoside transporters, respectively.
    Keywords:  Aberrant glycosylation; MUC4; Nucleoside transporters; Pancreatic cancer; Tn-MUC4 antibody
  30. Mol Cancer Res. 2021 Jan 26. pii: molcanres.0879.2020. [Epub ahead of print]
    Garnett S, de Bruyns A, Provencher-Tom V, Dutchak K, Shu R, Dankort D.
      Cellular senescence is characterized by a prolonged and predominantly irreversible cell cycle arrest state that is linked to loss of tissue function and aging in mammals. Moreover, in response to aberrant oncogenic signals such as those from oncogenic RAS or BRAF, senescence functions as an intrinsic tumor suppressor mechanism restraining tumor progression. In addition to this durable proliferative block, senescent cells adopt altered morphologies, transcriptional profiles and metabolism, while often possessing unusual heterochromatin formation termed senescence associated heterochromatic foci (SAHF). To uncover genes that are required to permit proliferation in the face of sustained oncogene signaling, we conducted an shRNA-based genetic screen in primary cells expressing inducible BRAF. Here, we show that depletion of a known glycolysis regulator, IAPP (islet amylin polypeptide also known as amylin), prevents RAS and BRAF oncogene-induced senescence (OIS) in human cells. Importantly, depletion of IAPP, resulted in changes of the cells' metabolome and this metabolic reprogramming was associated with widespread alterations in chromatin modifications compared to senescent cells. Conversely exogenous treatment of IAPP depleted cells with amylin restored OIS. Together, our results demonstrate that the metabolic regulator IAPP is important regulator of oncogene induced senescence. Moreover, they suggest that IAPP analog treatment or activation of IAPP signaling in RAS/BRAF mutant tumors may have therapeutic potential through senescence induction. Implications: These findings demonstrate that IAPP is a novel metabolic regulator of oncogene induce senescence and use of IAPP analogs may be therapeutically effective to restore growth arrest to BRAF and/or RAS mutant cancers.
  31. J Cell Sci. 2021 Jan 25. pii: jcs247874. [Epub ahead of print]134(2):
    Paci G, Caria J, Lemke EA.
      Bidirectional transport of macromolecules across the nuclear envelope is a hallmark of eukaryotic cells, in which the genetic material is compartmentalized inside the nucleus. The nuclear pore complex (NPC) is the major gateway to the nucleus and it regulates nucleocytoplasmic transport, which is key to processes including transcriptional regulation and cell cycle control. Accordingly, components of the nuclear transport machinery are often found to be dysregulated or hijacked in diseases. In this Cell Science at a Glance article and accompanying poster, we provide an overview of our current understanding of cargo transport through the NPC, from the basic transport signals and machinery to more emerging aspects, all from a 'cargo perspective'. Among these, we discuss the transport of large cargoes (>15 nm), as well as the roles of different cargo properties to nuclear transport, from size and number of bound nuclear transport receptors (NTRs), to surface and mechanical properties.
    Keywords:  Gene therapy; Large cargoes; Multivalent binding; Nuclear pore complex; Nuclear transport; Nuclear transport receptors; Virus
  32. Commun Biol. 2021 Jan 29. 4(1): 142
    Rackley B, Seong CS, Kiely E, Parker RE, Rupji M, Dwivedi B, Heddleston JM, Giang W, Anthony N, Chew TL, Gilbert-Ross M.
      The genetic and metabolic heterogeneity of RAS-driven cancers has confounded therapeutic strategies in the clinic. To address this, rapid and genetically tractable animal models are needed that recapitulate the heterogeneity of RAS-driven cancers in vivo. Here, we generate a Drosophila melanogaster model of Ras/Lkb1 mutant carcinoma. We show that low-level expression of oncogenic Ras (RasLow) promotes the survival of Lkb1 mutant tissue, but results in autonomous cell cycle arrest and non-autonomous overgrowth of wild-type tissue. In contrast, high-level expression of oncogenic Ras (RasHigh) transforms Lkb1 mutant tissue resulting in lethal malignant tumors. Using simultaneous multiview light-sheet microcopy, we have characterized invasion phenotypes of Ras/Lkb1 tumors in living larvae. Our molecular analysis reveals sustained activation of the AMPK pathway in malignant Ras/Lkb1 tumors, and demonstrate the genetic and pharmacologic dependence of these tumors on CaMK-activated Ampk. We further show that LKB1 mutant human lung adenocarcinoma patients with high levels of oncogenic KRAS exhibit worse overall survival and increased AMPK activation. Our results suggest that high levels of oncogenic KRAS is a driving event in the malignant transformation of LKB1 mutant tissue, and uncovers a vulnerability that may be used to target this aggressive genetic subset of RAS-driven tumors.
  33. Cancer Cell. 2021 Jan 27. pii: S1535-6108(21)00054-4. [Epub ahead of print]
    Muthuswamy SK.
      Pathologists use histological features to classify tumors and assign site of origin for metastasis. How and why tumors organize the way they do and recreate their histological organization during metastasis is unknown. Here, I discuss the concept of "histostasis" conferring tumors a histological memory and hypothesize its implications for metastasis.
  34. Proc Natl Acad Sci U S A. 2021 Feb 02. pii: e2020478118. [Epub ahead of print]118(5):
    Wijshake T, Zou Z, Chen B, Zhong L, Xiao G, Xie Y, Doench JG, Bennett L, Levine B.
      Beclin 1, an autophagy and haploinsufficient tumor-suppressor protein, is frequently monoallelically deleted in breast and ovarian cancers. However, the precise mechanisms by which Beclin 1 inhibits tumor growth remain largely unknown. To address this question, we performed a genome-wide CRISPR/Cas9 screen in MCF7 breast cancer cells to identify genes whose loss of function reverse Beclin 1-dependent inhibition of cellular proliferation. Small guide RNAs targeting CDH1 and CTNNA1, tumor-suppressor genes that encode cadherin/catenin complex members E-cadherin and alpha-catenin, respectively, were highly enriched in the screen. CRISPR/Cas9-mediated knockout of CDH1 or CTNNA1 reversed Beclin 1-dependent suppression of breast cancer cell proliferation and anchorage-independent growth. Moreover, deletion of CDH1 or CTNNA1 inhibited the tumor-suppressor effects of Beclin 1 in breast cancer xenografts. Enforced Beclin 1 expression in MCF7 cells and tumor xenografts increased cell surface localization of E-cadherin and decreased expression of mesenchymal markers and beta-catenin/Wnt target genes. Furthermore, CRISPR/Cas9-mediated knockout of BECN1 and the autophagy class III phosphatidylinositol kinase complex 2 (PI3KC3-C2) gene, UVRAG, but not PI3KC3-C1-specific ATG14 or other autophagy genes ATG13, ATG5, or ATG7, resulted in decreased E-cadherin plasma membrane and increased cytoplasmic E-cadherin localization. Taken together, these data reveal previously unrecognized cooperation between Beclin 1 and E-cadherin-mediated tumor suppression in breast cancer cells.
    Keywords:  Beclin 1; E-cadherin; breast cancer
  35. Nat Commun. 2021 Jan 29. 12(1): 694
    Masalmeh RHA, Taglini F, Rubio-Ramon C, Musialik KI, Higham J, Davidson-Smith H, Kafetzopoulos I, Pawlicka KP, Finan HM, Clark R, Wills J, Finch AJ, Murphy L, Sproul D.
      The aberrant gain of DNA methylation at CpG islands is frequently observed in colorectal tumours and may silence the expression of tumour suppressors such as MLH1. Current models propose that these CpG islands are targeted by de novo DNA methyltransferases in a sequence-specific manner, but this has not been tested. Using ectopically integrated CpG islands, here we find that aberrantly methylated CpG islands are subject to low levels of de novo DNA methylation activity in colorectal cancer cells. By delineating DNA methyltransferase targets, we find that instead de novo DNA methylation activity is targeted primarily to CpG islands marked by the histone modification H3K36me3, a mark associated with transcriptional elongation. These H3K36me3 marked CpG islands are heavily methylated in colorectal tumours and the normal colon suggesting that de novo DNA methyltransferase activity at CpG islands in colorectal cancer is focused on similar targets to normal tissues and not greatly remodelled by tumourigenesis.
  36. Genome Biol. 2021 Jan 29. 22(1): 54
    Li L, Ugalde AP, Scheele CLGJ, Dieter SM, Nagel R, Ma J, Pataskar A, Korkmaz G, Elkon R, Chien MP, You L, Su PR, Bleijerveld OB, Altelaar M, Momchev L, Manber Z, Han R, van Breugel PC, Lopes R, Ten Dijke P, van Rheenen J, Agami R.
      BACKGROUND: Frequent activation of the co-transcriptional factor YAP is observed in a large number of solid tumors. Activated YAP associates with enhancer loci via TEAD4-DNA-binding protein and stimulates cancer aggressiveness. Although thousands of YAP/TEAD4 binding-sites are annotated, their functional importance is unknown. Here, we aim at further identification of enhancer elements that are required for YAP functions.RESULTS: We first apply genome-wide ChIP profiling of YAP to systematically identify enhancers that are bound by YAP/TEAD4. Next, we implement a genetic approach to uncover functions of YAP/TEAD4-associated enhancers, demonstrate its robustness, and use it to reveal a network of enhancers required for YAP-mediated proliferation. We focus on EnhancerTRAM2, as its target gene TRAM2 shows the strongest expression-correlation with YAP activity in nearly all tumor types. Interestingly, TRAM2 phenocopies the YAP-induced cell proliferation, migration, and invasion phenotypes and correlates with poor patient survival. Mechanistically, we identify FSTL-1 as a major direct client of TRAM2 that is involved in these phenotypes. Thus, TRAM2 is a key novel mediator of YAP-induced oncogenic proliferation and cellular invasiveness.
    CONCLUSIONS: YAP is a transcription co-factor that binds to thousands of enhancer loci and stimulates tumor aggressiveness. Using unbiased functional approaches, we dissect YAP enhancer network and characterize TRAM2 as a novel mediator of cellular proliferation, migration, and invasion. Our findings elucidate how YAP induces cancer aggressiveness and may assist diagnosis of cancer metastasis.
    Keywords:  Enhancer; Gene regulation; Invasion; Migration; Proliferation; TRAM2; YAP
  37. Nat Commun. 2021 01 27. 12(1): 614
    Lita A, Pliss A, Kuzmin A, Yamasaki T, Zhang L, Dowdy T, Burks C, de Val N, Celiku O, Ruiz-Rodado V, Nicoli ER, Kruhlak M, Andresson T, Das S, Yang C, Schmitt R, Herold-Mende C, Gilbert MR, Prasad PN, Larion M.
      Infiltrating gliomas are devastating and incurable tumors. Amongst all gliomas, those harboring a mutation in isocitrate dehydrogenase 1 mutation (IDH1mut) acquire a different tumor biology and clinical manifestation from those that are IDH1WT. Understanding the unique metabolic profile reprogrammed by IDH1 mutation has the potential to identify new molecular targets for glioma therapy. Herein, we uncover increased monounsaturated fatty acids (MUFA) and their phospholipids in endoplasmic reticulum (ER), generated by IDH1 mutation, that are responsible for Golgi and ER dilation. We demonstrate a direct link between the IDH1 mutation and this organelle morphology via D-2HG-induced stearyl-CoA desaturase (SCD) overexpression, the rate-limiting enzyme in MUFA biosynthesis. Inhibition of IDH1 mutation or SCD silencing restores ER and Golgi morphology, while D-2HG and oleic acid induces morphological defects in these organelles. Moreover, addition of oleic acid, which tilts the balance towards elevated levels of MUFA, produces IDH1mut-specific cellular apoptosis. Collectively, these results suggest that IDH1mut-induced SCD overexpression can rearrange the distribution of lipids in the organelles of glioma cells, providing new insight into the link between lipid metabolism and organelle morphology in these cells, with potential and unique therapeutic implications.
  38. Nat Rev Mol Cell Biol. 2021 Jan 28.
    Alberti S, Hyman AA.
      Biomolecular condensates are membraneless intracellular assemblies that often form via liquid-liquid phase separation and have the ability to concentrate biopolymers. Research over the past 10 years has revealed that condensates play fundamental roles in cellular organization and physiology, and our understanding of the molecular principles, components and forces underlying their formation has substantially increased. Condensate assembly is tightly regulated in the intracellular environment, and failure to control condensate properties, formation and dissolution can lead to protein misfolding and aggregation, which are often the cause of ageing-associated diseases. In this Review, we describe the mechanisms and regulation of condensate assembly and dissolution, highlight recent advances in understanding the role of biomolecular condensates in ageing and disease, and discuss how cellular stress, ageing-related loss of homeostasis and a decline in protein quality control may contribute to the formation of aberrant, disease-causing condensates. Our improved understanding of condensate pathology provides a promising path for the treatment of protein aggregation diseases.
  39. Nat Mater. 2021 Jan 25.
    Reuten R, Zendehroud S, Nicolau M, Fleischhauer L, Laitala A, Kiderlen S, Nikodemus D, Wullkopf L, Nielsen SR, McNeilly S, Prein C, Rafaeva M, Schoof EM, Furtwängler B, Porse BT, Kim H, Won KJ, Sudhop S, Zornhagen KW, Suhr F, Maniati E, Pearce OMT, Koch M, Oddershede LB, Van Agtmael T, Madsen CD, Mayorca-Guiliani AE, Bloch W, Netz RR, Clausen-Schaumann H, Erler JT.
      The basement membrane (BM) is a special type of extracellular matrix and presents the major barrier cancer cells have to overcome multiple times to form metastases. Here we show that BM stiffness is a major determinant of metastases formation in several tissues and identify netrin-4 (Net4) as a key regulator of BM stiffness. Mechanistically, our biophysical and functional analyses in combination with mathematical simulations show that Net4 softens the mechanical properties of native BMs by opening laminin node complexes, decreasing cancer cell potential to transmigrate this barrier despite creating bigger pores. Our results therefore reveal that BM stiffness is dominant over pore size, and that the mechanical properties of 'normal' BMs determine metastases formation and patient survival independent of cancer-mediated alterations. Thus, identifying individual Net4 protein levels within native BMs in major metastatic organs may have the potential to define patient survival even before tumour formation. The ratio of Net4 to laminin molecules determines BM stiffness, such that the more Net4, the softer the BM, thereby decreasing cancer cell invasion activity.
  40. Nat Genet. 2021 Jan 25.
    Jay P, Chouteau M, Whibley A, Bastide H, Parrinello H, Llaurens V, Joron M.
      Chromosomal inversions are ubiquitous in genomes and often coordinate complex phenotypes, such as the covariation of behavior and morphology in many birds, fishes, insects or mammals1-11. However, why and how inversions become associated with polymorphic traits remains obscure. Here we show that despite a strong selective advantage when they form, inversions accumulate recessive deleterious mutations that generate frequency-dependent selection and promote their maintenance at intermediate frequency. Combining genomics and in vivo fitness analyses in a model butterfly for wing-pattern polymorphism, Heliconius numata, we reveal that three ecologically advantageous inversions have built up a heavy mutational load from the sequential accumulation of deleterious mutations and transposable elements. Inversions associate with sharply reduced viability when homozygous, which prevents them from replacing ancestral chromosome arrangements. Our results suggest that other complex polymorphisms, rather than representing adaptations to competing ecological optima, could evolve because chromosomal rearrangements are intrinsically prone to carrying recessive harmful mutations.
  41. EMBO J. 2021 Jan 25. e107525
    Krenning L, Raaijmakers JA, Medema RH.
      Accurate control of centrosome number is essential for proper chromosome segregation, and it is well established that centrosome abnormalities can trigger a p53-dependent cell cycle arrest. Two new studies published in The EMBO Journal demonstrate how PIDD1 is recruited to centrosomes and that the localization of PIDD1 to distal appendages of centrosomes is required for PIDDosome activation at clustered supernumerary centrosomes.
  42. Nat Commun. 2021 01 28. 12(1): 646
    Matsumoto T, Wakefield L, Peters A, Peto M, Spellman P, Grompe M.
      Polyploidy is a hallmark of cancer, and closely related to chromosomal instability involved in cancer progression. Importantly, polyploid cells also exist in some normal tissues. Polyploid hepatocytes proliferate and dynamically reduce their ploidy during liver regeneration. This raises the question whether proliferating polyploids are prone to cancer via chromosome missegregation during mitosis and/or ploidy reduction. Conversely polyploids could be resistant to tumor development due to their redundant genomes. Therefore, the tumor-initiation risk of physiologic polyploidy and ploidy reduction is still unclear. Using in vivo lineage tracing we here show that polyploid hepatocytes readily form liver tumors via frequent ploidy reduction. Polyploid hepatocytes give rise to regenerative nodules with chromosome aberrations, which are enhanced by ploidy reduction. Although polyploidy should theoretically prevent tumor suppressor loss, the high frequency of ploidy reduction negates this protection. Importantly, polyploid hepatocytes that undergo multiple rounds of cell division become predominantly mononucleated and are resistant to ploidy reduction. Our results suggest that ploidy reduction is an early step in the initiation of carcinogenesis from polyploid hepatocytes.
  43. Nat Chem Biol. 2021 Jan 28.
    Hill SM, Wrobel L, Ashkenazi A, Fernandez-Estevez M, Tan K, Bürli RW, Rubinsztein DC.
      Autophagy is an essential cellular process that removes harmful protein species, and autophagy upregulation may be able to protect against neurodegeneration and various pathogens. Here, we have identified the essential protein VCP/p97 (VCP, valosin-containing protein) as a novel regulator of autophagosome biogenesis, where VCP regulates autophagy induction in two ways, both dependent on Beclin-1. Utilizing small-molecule inhibitors of VCP ATPase activity, we show that VCP stabilizes Beclin-1 levels by promoting the deubiquitinase activity of ataxin-3 towards Beclin-1. VCP also regulates the assembly and activity of the Beclin-1-containing phosphatidylinositol-3-kinase (PI3K) complex I, thus regulating the production of PI(3)P, a key signaling lipid responsible for the recruitment of downstream autophagy factors. A decreased level of VCP, or inhibition of its ATPase activity, impairs starvation-induced production of PI(3)P and limits downstream recruitment of WIPI2, ATG16L and LC3, thereby decreasing autophagosome formation, illustrating an important role for VCP in early autophagy initiation.
  44. Nat Metab. 2021 Jan 28.
    Geeraerts SL, Heylen E, De Keersmaecker K, Kampen KR.
      Cancer cells reprogramme their metabolism to support unrestrained proliferation and survival in nutrient-poor conditions. Whereas non-transformed cells often have lower demands for serine and glycine, several cancer subtypes hyperactivate intracellular serine and glycine synthesis and become addicted to de novo production. Copy-number amplifications of serine- and glycine-synthesis genes and genetic alterations in common oncogenes and tumour-suppressor genes enhance serine and glycine synthesis, resulting in high production and secretion of these oncogenesis-supportive metabolites. In this Review, we discuss the contribution of serine and glycine synthesis to cancer progression. By relying on de novo synthesis pathways, cancer cells are able to enhance macromolecule synthesis, neutralize high levels of oxidative stress and regulate methylation and tRNA formylation. Furthermore, we discuss the immunosuppressive potential of serine and glycine, and the essentiality of both amino acids to promoting survival of non-transformed neighbouring cells. Finally, we point to the emerging data proposing moonlighting functions of serine- and glycine-synthesis enzymes and examine promising small molecules targeting serine and glycine synthesis.
  45. J Cell Sci. 2021 Jan 25. pii: jcs254599. [Epub ahead of print]134(2):
    Kerjouan A, Boyault C, Oddou C, Hiriart-Bryant E, Grichine A, Kraut A, Pezet M, Balland M, Faurobert E, Bonnet I, Coute Y, Fourcade B, Albiges-Rizo C, Destaing O.
      Upon activation by different transmembrane receptors, the same signaling protein can induce distinct cellular responses. A way to decipher the mechanisms of such pleiotropic signaling activity is to directly manipulate the decision-making activity that supports the selection between distinct cellular responses. We developed an optogenetic probe (optoSRC) to control SRC signaling, an example of a pleiotropic signaling node, and we demonstrated its ability to generate different acto-adhesive structures (lamellipodia or invadosomes) upon distinct spatio-temporal control of SRC kinase activity. The occurrence of each acto-adhesive structure was simply dictated by the dynamics of optoSRC nanoclusters in adhesive sites, which were dependent on the SH3 and Unique domains of the protein. The different decision-making events regulated by optoSRC dynamics induced distinct downstream signaling pathways, which we characterized using time-resolved proteomic and network analyses. Collectively, by manipulating the molecular mobility of SRC kinase activity, these experiments reveal the pleiotropy-encoding mechanism of SRC signaling.
    Keywords:  Adhesions; Conformational intermediates; Cytoskeletal; Encoding intracellular signaling; Invasion; Migration; Optogenetics; Pleiotropy; SRC; Time-resolved phosphoproteomics
  46. Nat Commun. 2021 Jan 29. 12(1): 707
    Yin Z, Burger N, Kula-Alwar D, Aksentijević D, Bridges HR, Prag HA, Grba DN, Viscomi C, James AM, Mottahedin A, Krieg T, Murphy MP, Hirst J.
      Mitochondrial complex I is central to the pathological reactive oxygen species (ROS) production that underlies cardiac ischemia-reperfusion (IR) injury. ND6-P25L mice are homoplasmic for a disease-causing mtDNA point mutation encoding the P25L substitution in the ND6 subunit of complex I. The cryo-EM structure of ND6-P25L complex I revealed subtle structural changes that facilitate rapid conversion to the "deactive" state, usually formed only after prolonged inactivity. Despite its tendency to adopt the "deactive" state, the mutant complex is fully active for NADH oxidation, but cannot generate ROS by reverse electron transfer (RET). ND6-P25L mitochondria function normally, except for their lack of RET ROS production, and ND6-P25L mice are protected against cardiac IR injury in vivo. Thus, this single point mutation in complex I, which does not affect oxidative phosphorylation but renders the complex unable to catalyse RET, demonstrates the pathological role of ROS production by RET during IR injury.
  47. Dev Cell. 2021 Jan 25. pii: S1534-5807(20)30977-1. [Epub ahead of print]56(2): 213-226
    Shamipour S, Caballero-Mancebo S, Heisenberg CP.
      Cytoplasm is a gel-like crowded environment composed of various macromolecules, organelles, cytoskeletal networks, and cytosol. The structure of the cytoplasm is highly organized and heterogeneous due to the crowding of its constituents and their effective compartmentalization. In such an environment, the diffusive dynamics of the molecules are restricted, an effect that is further amplified by clustering and anchoring of molecules. Despite the crowded nature of the cytoplasm at the microscopic scale, large-scale reorganization of the cytoplasm is essential for important cellular functions, such as cell division and polarization. How such mesoscale reorganization of the cytoplasm is achieved, especially for large cells such as oocytes or syncytial tissues that can span hundreds of micrometers in size, is only beginning to be understood. In this review, we will discuss recent advances in elucidating the molecular, cellular, and biophysical mechanisms by which the cytoskeleton drives cytoplasmic reorganization across different scales, structures, and species.
  48. Semin Cancer Biol. 2021 Jan 21. pii: S1044-579X(21)00010-9. [Epub ahead of print]
    Gal H, Majewska J, Krizhanovsky V.
      Cellular senescence, a stable form of cell cycle arrest, accompanied by pronounced secretory activity, has functional roles in both physiological and pathological conditions. Although senescence has been linked for a long time with cancer and ageing, recent studies have revealed a functional role of senescence in development, regeneration and reprogramming. Notably, the transient presence of senescent cells may be beneficial, in contrast to the potential deleterious effects of persistent senescence in aged or chronically damaged tissues. We will discuss how senescence contributes to embryonic development, cell plasticity and tissue regeneration, as a highly coordinated and programmed cellular state.
    Keywords:  Cellular senescence; Embryonic development; Placenta; Regeneration
  49. FASEB J. 2021 Feb;35(2): e21365
    Duran-Güell M, Flores-Costa R, Casulleras M, López-Vicario C, Titos E, Díaz A, Alcaraz-Quiles J, Horrillo R, Costa M, Fernández J, Arroyo V, Clària J.
      Besides its oncotic power, albumin exerts pleiotropic actions, including binding, transport, and detoxification of endogenous and exogenous molecules, antioxidant activity, and modulation of immune and inflammatory responses. In particular, recent studies have demonstrated that albumin reduces leukocyte cytokine production. Here, we investigated whether albumin also has the ability to protect tissues from the damaging actions of these inflammatory mediators. We circumscribed our investigation to tumor necrosis factor (TNF) α, which exemplifies the connection between immunity and tissue injury. In vivo experiments in analbuminemic mice showed that these mice exhibit a more pronounced response to a model of TNFα-mediated liver injury induced by the administration of lipopolysaccharide (LPS) and D-galactosamine (D-gal). A tissue protective action against LPS/D-gal liver injury was also observed during the administration of human albumin to humanized mice expressing the human genes for albumin and neonatal Fc receptor (hAlb+/+ /hFcRn+/+ ) with preestablished carbon tetrachloride (CCl4 )-induced early cirrhosis. The cytoprotective actions of albumin against TNFα-induced injury were confirmed ex vivo, in precision-cut liver slices, and in vitro, in primary hepatocytes in culture. Albumin protective actions were independent of its scavenging properties and were reproduced by recombinant human albumin expressed in Oryza sativa. Albumin cytoprotection against TNFα injury was related to inhibition of lysosomal cathepsin B leakage accompanied by reductions in mitochondrial cytochrome c release and caspase-3 activity. These data provide evidence that in addition to reducing cytokines, the albumin molecule also has the ability to protect tissues against inflammatory injury.
    Keywords:  albumin; cytokine-induced tissue injury; liver cells; liver injury; mitochondrial damage
  50. Dev Cell. 2021 Jan 25. pii: S1534-5807(21)00033-2. [Epub ahead of print]56(2): 156-158
    Long JT, Lammerding J.
      How cells sense their physical microenvironment remains incompletely understood. In two recent Science articles, Lomakin et al. (2020) and Venturini et al. (2020) demonstrate that progressive nuclear deformation associated with cellular confinement triggers intracellular events that promote cell contractility and migration, revealing the nucleus to serve as a central mechanosensor.
  51. Nat Commun. 2021 Jan 29. 12(1): 704
    Vaughan CA, Singh S, Subler MA, Windle JJ, Inoue K, Fry EA, Pillappa R, Grossman SR, Windle B, Andrew Yeudall W, Deb SP, Deb S.
      p53 mutations with single amino acid changes in cancer often lead to dominant oncogenic changes. Here, we have developed a mouse model of gain-of-function (GOF) p53-driven lung cancer utilizing conditionally active LSL p53-R172H and LSL K-Ras-G12D knock-in alleles that can be activated by Cre in lung club cells. Mutation of the p53 transactivation domain (TAD) (p53-L25Q/W26S/R172H) eliminating significant transactivation activity resulted in loss of tumorigenicity, demonstrating that transactivation mediated by or dependent on TAD is required for oncogenicity by GOF p53. GOF p53 TAD mutations significantly reduce phosphorylation of nearby p53 serine 20 (S20), which is a target for PLK3 phosphorylation. Knocking out PLK3 attenuated S20 phosphorylation along with transactivation and oncogenicity by GOF p53, indicating that GOF p53 exploits PLK3 to trigger its transactivation capability and exert oncogenic functions. Our data show a mechanistic involvement of PLK3 in mutant p53 pathway of oncogenesis.
  52. STAR Protoc. 2021 Mar 19. 2(1): 100272
    Skruber K, Read TA, Vitriol EA.
      Here, we detail a protocol using electroporation to precisely deliver defined amounts of purified protein into CAD cells. This method allows one million cells to be electroporated with protein simultaneously, with high delivery efficiency and low cell death. Further, by circumventing the normal biosynthetic pathway, proteins can be studied without the complication of post-translational modifications and before a transcriptional response can be initiated. This protocol will be useful for any researcher who is interested in protein concentration-dependent cellular phenotypes. For complete details on the use and execution of this protocol, please refer to Skruber et al. (2020).
    Keywords:  Cell biology; Cell culture; Cell-based assays; Microscopy; Molecular/chemical probes; Protein expression and purification
  53. Mol Metab. 2021 Jan 20. pii: S2212-8778(21)00009-0. [Epub ahead of print] 101169
    Lemmer IL, Willemsen N, Hilal N, Bartelt A.
      BACKGROUND: The global rise of metabolic disorders, such as obesity, diabetes type 2 and cardiovascular disease, demands a thorough molecular understanding of the cellular mechanisms that govern health or disease. The endoplasmic reticulum (ER) is a key organelle for cellular function and metabolic adaptation and, therefore, disturbed ER function, "ER stress", is a key feature of metabolic disorders.SCOPE OF REVIEW: As ER stress remains an ill-defined phenomenon, this review provides a general guide to understanding the nature, aetiology and consequences of ER stress in metabolic disorders. We define ER stress by its type of stressor, which is driven by proteotoxicity, lipotoxicity, and/or glucotoxicity. We discuss the implications of ER stress in metabolic disorders by reviewing evidence implicating ER phenotypes and organelle communication, protein quality control, calcium homeostasis, lipid and carbohydrate metabolism, and inflammation as key mechanisms in the development of ER stress and metabolic dysfunction.
    MAJOR CONCLUSIONS: In mammalian biology, ER is a phenotypically and functionally diverse platform for nutrient sensing, which is critical for cell-type specific metabolic control by e.g. hepatocytes, adipocytes, muscle cells, and neurons. In these cells, ER stress is a distinct, transient state of functional imbalance, which is usually resolved by the activation of adaptive programs such as the unfolded protein response (UPR), ER-associated protein degradation (ERAD), or autophagy. However, challenges to proteostasis also impact lipid and glucose metabolism and vice versa. In the ER, both sensing and adaptive measures are integrated and failure of the ER to adapt leads to aberrant metabolism, organelle dysfunction, insulin resistance, and inflammation. In conclusion, the ER is intricately linked to a wide spectrum of cellular functions and is a critical component in maintaining and restoring metabolic health.
    Keywords:  ERAD; NFE2L1; Obesity; UPR; UPS; autophagy; calcium homeostasis; diabetes; endoplasmic reticulum; glucotoxicity; inflammation; lipid metabolism; lipotoxicity; proteostasis; proteotoxicity
  54. Nat Rev Mol Cell Biol. 2021 Jan 27.
    Canovas B, Nebreda AR.
      The ability of cells to deal with different types of stressful situations in a precise and coordinated manner is key for survival and involves various signalling networks. Over the past 25 years, p38 kinases - in particular, p38α - have been implicated in the cellular response to stress at many levels. These span from environmental and intracellular stresses, such as hyperosmolarity, oxidative stress or DNA damage, to physiological situations that involve important cellular changes such as differentiation. Given that p38α controls a plethora of functions, dysregulation of this pathway has been linked to diseases such as inflammation, immune disorders or cancer, suggesting the possibility that targeting p38α could be of therapeutic interest. In this Review, we discuss the organization of this signalling pathway focusing on the diversity of p38α substrates, their mechanisms and their links to particular cellular functions. We then address how the different cellular responses can be generated depending on the signal received and the cell type, and highlight the roles of this kinase in human physiology and in pathological contexts.
  55. Eur J Clin Nutr. 2021 Jan 29.
    Shannon OM, Ashor AW, Scialo F, Saretzki G, Martin-Ruiz C, Lara J, Matu J, Griffiths A, Robinson N, Lillà L, Stevenson E, Stephan BCM, Minihane AM, Siervo M, Mathers JC.
      Ageing is a multifactorial process associated with reduced function and increased risk of morbidity and mortality. Recently, nine cellular and molecular hallmarks of ageing have been identified, which characterise the ageing process, and collectively, may be key determinants of the ageing trajectory. These include genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion and altered intercellular communication. Healthier dietary patterns reduce the risk of age-related diseases and increase longevity and may influence positively one or more of these hallmarks. The Mediterranean dietary pattern (MedDiet) is a plant-based eating pattern that was typical of countries such as Greece, Spain, and Italy pre-globalisation of the food system and which is associated with better health during ageing. Here we review the potential effects of a MedDiet on each of the nine hallmarks of ageing, and provide evidence that the MedDiet as a whole, or individual elements of this dietary pattern, may influence each hallmark positively-effects which may contribute to the beneficial effects of this dietary pattern on age-related disease risk and longevity. We also highlight potential avenues for future research.
  56. Elife. 2021 Jan 26. pii: e64004. [Epub ahead of print]10
    Dutagaci B, Nawrocki G, Goodluck J, Ashkarran AA, Hoogstraten CG, Lapidus LJ, Feig M.
      Phase separation processes are increasingly being recognized as important organizing mechanisms of biological macromolecules in cellular environments. Well established drivers of phase separation are multi-valency and intrinsic disorder. Here, we show that globular macromolecules may condense simply based on electrostatic complementarity. More specifically, phase separation of mixtures between RNA and positively charged proteins is described from a combination of multiscale computer simulations with microscopy and spectroscopy experiments. Phase diagrams were mapped out as a function of molecular concentrations in experiment and as a function of molecular size and temperature via simulations. The resulting condensates were found to retain at least some degree of internal dynamics varying as a function of the molecular composition. The results suggest a more general principle for phase separation that is based primarily on electrostatic complementarity without invoking polymer properties as in most previous studies. Simulation results furthermore suggest that such phase separation may occur widely in heterogenous cellular environment between nucleic acid and protein components.
    Keywords:  none; physics of living systems
  57. Front Oncol. 2020 ;10 575264
    Liu R, Liao YZ, Zhang W, Zhou HH.
      Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with high heterogeneity and dismal survival rates. Tumor immune microenvironment plays a critical role in sensitive to chemotherapy and prognosis. Herein, we determined the relevance of the composition of tumor-infiltrating immune cells to clinical outcomes in PDACs, and we evaluated these effects by molecular subtype.Experimental Design: Data of 1,274 samples from publically available datasets were collected. Molecular subtypes were predicted with support vector machine. Twenty-two subsets of immune cells were estimated with CIBERSORTx. The associations between each cell subset and overall survival (OS), relapse free survival (RFS), and complete response (CR) to chemotherapy were evaluated, modelling cellular proportions as quartiles.
    Results: An immune-related cluster was identified with unsupervised hierarchical clustering of hallmark pathways. Of the immune cells investigated, M0 macrophages emerged as closely associated with worse OS (HR =1.23, 95% CI = 1.15-1.31, p=1.57×10-9) and RFS (HR = 1.14, 95% CI =1.04-1.25, p=2.93×10-3), regardless of molecular subtypes. The CD8+ T cells conferred favorable survival. The neutrophils conferred poor OS overall (HR=1.17, 95% CI=1.10-1.23, p=1.74×10-7) and within the classical subtype. In the basal-like subtype, activated mast cells were associated with worse OS. Consensus clustering revealed six immune subgroups with distinct survival patterns and CR rates. The higher expression of PD1 was associated with better OS.
    Conclusions: The immune cellular composition infiltrate in PDAC are likely to have effects on prognosis. Further exploration of the cellular immune response has the potential to identify candidates for immunotherapy.
    Keywords:  M0 macrophages; overall survival; pancreatic ductal adenocarcinoma; relapse free survival; tumor-immune infiltration
  58. Cancer Immunol Res. 2021 Jan 26. pii: canimm.0317.2020. [Epub ahead of print]
    Badeaux MD, Rolig AS, Agnello G, Enzler D, Kasiewicz MJ, Priddy L, Wiggins JF, Muir A, Sullivan MR, Van Cleef J, Daige C, Vander Heiden MG, Rajamanickam V, Wooldridge J, Redmond WL, Rowlinson SW.
      Metabolic dysregulation is a hallmark of cancer; for instance, many tumors exhibit auxotrophy for various amino acids, such as arginine, as they are unable to meet the demand for these amino acids through endogenous production. This vulnerability can be exploited by employing therapeutic strategies that deplete systemic arginine in order to limit the growth and survival of arginine auxotrophic tumors. Pegzilarginase, a human arginase 1 enzyme engineered to have superior stability and enzymatic activity relative to the native human arginase 1 enzyme, depletes systemic arginine by converting it to ornithine and urea. Therapeutic administration of pegzilarginase in the setting of arginine auxotrophic tumors exerts direct anti-tumor activity by starving the tumor of exogenous arginine. We hypothesized that in addition to this direct effect, pegzilarginase treatment indirectly augments anti-tumor immunity through increased antigen presentation, thus making pegzilarginase a prime candidate for combination therapy with immuno-oncology (I-O) agents. Indeed, tumor-bearing mice (CT26; MC38; MCA-205) receiving pegzilarginase in combination with aPD-L1 or agonist aOX40 mAb experienced significantly increased survival relative to animals receiving I-O monotherapy. Combination pegzilarginase/immunotherapy induced robust anti-tumor immunity characterized by increased intratumoral effector CD8+ T cells and M1-polarization of tumor-associated macrophages. Our data suggests potential mechanisms of synergy between pegzilarginase and I-O agents that include increased intratumoral MHC expression on both APCs and tumor cells, and increased presence of M1-like anti-tumor macrophages. These data support the clinical evaluation of I-O agents in conjunction with pegzilarginase for the treatment of patients with cancer.
  59. Am J Physiol Gastrointest Liver Physiol. 2021 Jan 27.
    Li C, Peng Z, Wang Y, Lam G, Nissen N, Tang J, Yuan X, Lewis M, Greene MI, Pandol SJ, Wang Q.
      Pancreatic ductal adenocarcinoma (PDAC) is highly metastatic and represents one of the deadliest forms of human cancers. Previous studies showed that activation of Yes-associated protein 1 (YAP1) plays a key role in malignant transformation in the pancreas. In this study, we found that YAP1 regulates the expression of epithelial cell transforming 2 (ECT2), a guanine nucleotide exchange factor for Rho-like GTPases. By immunohistochemistry analysis of human tissues, we show that ECT2 is highly expressed in primary PDAC and liver metastasis but not in normal pancreas. These correlations were also observed in a mouse model of PDAC, where pancreatic transformation is driven by mutants of Kras and Trp53. Notably, nuclear ECT2 is upregulated in the transition from pre-neoplastic lesions to PDAC. High levels of YAP1 or ECT2 expression correlates with poor overall survival rate of PDAC patients. We further demonstrate that ECT2 is required for pancreatic cancer cell proliferation and migration in vitro. Finally, using a syngeneic orthotopic xenograft mouse model for pancreatic cancer, we found that ablation of ECT2 expression reduces pancreatic cancer growth and dissemination to the liver. These findings highlight a critical role of ECT2 in promoting pancreatic cancer growth and metastasis and provides insights into development of novel methods for early detection and treatment.
    Keywords:  ECT2; IL11; Pancreatic cancer; YAP1; metastasis
  60. Trends Genet. 2021 Jan 22. pii: S0168-9525(20)30336-X. [Epub ahead of print]
    Guo Y, Zhao S, Wang GG.
      Modulation of chromatin structure and/or modification by Polycomb repressive complexes (PRCs) provides an important means to partition the genome into functionally distinct subdomains and to regulate the activity of the underlying genes. Both the enzymatic activity of PRC2 and its chromatin recruitment, spreading, and eviction are exquisitely regulated via interactions with cofactors and DNA elements (such as unmethylated CpG islands), histones, RNA (nascent mRNA and long noncoding RNA), and R-loops. PRC2-catalyzed histone H3 lysine 27 trimethylation (H3K27me3) is recognized by distinct classes of effectors such as canonical PRC1 and BAH module-containing proteins (notably BAHCC1 in human). These effectors mediate gene silencing by different mechanisms including phase separation-related chromatin compaction and histone deacetylation. We discuss recent advances in understanding the structural architecture of PRC2, the regulation of its activity and chromatin recruitment, and the molecular mechanisms underlying Polycomb-mediated gene silencing. Because PRC deregulation is intimately associated with the development of diseases, a better appreciation of Polycomb-based (epi)genomic regulation will have far-reaching implications in biology and medicine.
    Keywords:  BAH; CpG island; H3K27me3; PRC1; PRC2; Polycomb; R-loop; RNA; allosteric activation; chromatin; chromodomain; histone; methylation; phase separation; transcription
  61. Cancer Res. 2021 Jan 26. pii: canres.3020.2019. [Epub ahead of print]
    Barnabas GD, Sang Lee J, Shami T, Harel M, Beck L, Selitrennik M, Jerby-Arnon L, Erez N, Ruppin E, Geiger T.
      Cancer-specific metabolic phenotypes and their vulnerabilities represent a viable area of cancer research. In this study, we explored the association of breast cancer subtypes with different metabolic phenotypes and identified isocitrate dehydrogenase 2 (IDH2) as a key player in triple-negative breast cancer (TNBC) and HER2. Functional assays combined with mass spectrometry-based analyses revealed the oncogenic role of IDH2 in cell proliferation, anchorage-independent growth, glycolysis, mitochondrial respiration, and antioxidant defense. Genome-scale metabolic modeling identified PHGDH and PSAT1 as the synthetic dosage lethal (SDL) partners of IDH2. In agreement, CRISPR-Cas9 knockout of PHGDH and PSAT1 showed the essentiality of serine biosynthesis proteins in IDH2-high cells. The clinical significance of the SDL interaction was supported by patients with IDH2-high/PHGDH-low tumors, who exhibited longer survival than patients with IDH2-high/PHGDH-high tumors. Furthermore, PHGDH inhibitors were effective in treating IDH2-high cells in vitro and in vivo. Altogether, our study creates a new link between two known cancer regulators and emphasizes PHGDH as a promising target for TNBC with IDH2 overexpression.
  62. Trends Cancer. 2021 Jan 23. pii: S2405-8033(20)30342-3. [Epub ahead of print]
    Liu Z, Rabadan R.
      Most human genes undergo alternative splicing (AS), and dysregulation of alternative splicing contributes to tumor initiation and progression. Computational analysis of genomic and transcriptomic data enables the systematic characterization of alternative splicing and its functional role in cancer. In this review, we summarize the latest computational approaches to studying alternative splicing in cancer and the current limitations of the most popular tools in this field. Finally, we describe some of the current computational challenges in the characterization of the role of alternative splicing in cancer.
    Keywords:  alternative splicing; cancer; computational analysis; spliceosomal mutations
  63. Blood Adv. 2021 Jan 26. 5(2): 487-495
    Hisada Y, Garratt KB, Maqsood A, Grover SP, Kawano T, Cooley BC, Erlich J, Moik F, Flick MJ, Pabinger I, Mackman N, Ay C.
      Pancreatic cancer patients have a high risk of venous thromboembolism (VTE). Plasminogen activator inhibitor 1 (PAI-1) inhibits plasminogen activators and increases the risk of thrombosis. PAI-1 is expressed by pancreatic tumors and human pancreatic cell lines. However, to date, there are no studies analyzing the association of active PAI-1 and VTE in pancreatic cancer patients. We investigated the association of active PAI-1 in plasma and VTE in pancreatic cancer patients. In addition, we determined if the presence of human pancreatic tumors expressing PAI-1 impairs venous thrombus resolution in mice. Plasma levels of active PAI-1 in patients with pancreatic cancer and mice bearing human tumors were determined by enzyme-linked immunosorbent assay. We measured PAI-1 expression in 5 different human pancreatic cancer cell lines and found that PANC-1 cells expressed the highest level. PANC-1 tumors were grown in nude mice. Venous thrombosis was induced by complete ligation of the inferior vena cava (IVC). Levels of active PAI-1 were independently associated with increased risk of VTE in patients with pancreatic cancer (subdistribution hazard ratio per doubling of levels: 1.39 [95% confidence interval, 1.09-1.78], P = .007). Mice bearing PANC-1 tumors had increased levels of both active human and active mouse PAI-1 and decreased levels of plasmin activity. Importantly, mice bearing PANC-1 tumors exhibited impaired venous thrombus resolution 8 days after IVC stasis compared with nontumor controls. Our results suggest that PAI-1 contributes to VTE in pancreatic cancer.
  64. Nat Cell Biol. 2021 Jan 25.
    Baumgartner ME, Dinan MP, Langton PF, Kucinski I, Piddini E.
      Cell competition allows winner cells to eliminate less fit loser cells in tissues. In Minute cell competition, cells with a heterozygous mutation in ribosome genes, such as RpS3+/- cells, are eliminated by wild-type cells. How cells are primed as losers is partially understood and it has been proposed that reduced translation underpins the loser status of ribosome mutant, or Minute, cells. Here, using Drosophila, we show that reduced translation does not cause cell competition. Instead, we identify proteotoxic stress as the underlying cause of the loser status for Minute competition and competition induced by mahjong, an unrelated loser gene. RpS3+/- cells exhibit reduced autophagic and proteasomal flux, accumulate protein aggregates and can be rescued from competition by improving their proteostasis. Conversely, inducing proteotoxic stress is sufficient to turn otherwise wild-type cells into losers. Thus, we propose that tissues may preserve their health through a proteostasis-based mechanism of cell competition and cell selection.
  65. Science. 2021 Jan 29. pii: eaax2656. [Epub ahead of print]371(6528):
    Alon S, Goodwin DR, Sinha A, Wassie AT, Chen F, Daugharthy ER, Bando Y, Kajita A, Xue AG, Marrett K, Prior R, Cui Y, Payne AC, Yao CC, Suk HJ, Wang R, Yu CJ, Tillberg P, Reginato P, Pak N, Liu S, Punthambaker S, Iyer EPR, Kohman RE, Miller JA, Lein ES, Lako A, Cullen N, Rodig S, Helvie K, Abravanel DL, Wagle N, Johnson BE, Klughammer J, Slyper M, Waldman J, Jané-Valbuena J, Rozenblatt-Rosen O, Regev A, , Church GM, Marblestone AH, Boyden ES.
      Methods for highly multiplexed RNA imaging are limited in spatial resolution and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to the mouse brain, which yielded the readout of thousands of genes, including splice variants. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in the neurons of the mouse hippocampus, revealing patterns across multiple cell types, layer-specific cell types across the mouse visual cortex, and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus, ExSeq enables highly multiplexed mapping of RNAs from nanoscale to system scale.
  66. Clin Epigenetics. 2021 Jan 28. 13(1): 20
    Mattesen TB, Andersen CL, Bramsen JB.
      BACKGROUND: Transcriptional analysis is widely used to study the molecular biology of cancer and hold great biomarker potential for clinical patient stratification. Yet, accurate transcriptional profiling requires RNA of a high quality, which often cannot be retrieved from formalin-fixed, paraffin-embedded (FFPE) tumor tissue that is routinely collected and archived in clinical departments. To overcome this roadblock to clinical testing, we previously developed MethCORR, a method that infers gene expression from DNA methylation data, which is robustly retrieved from FFPE tissue. MethCORR was originally developed for colorectal cancer and with this study, we aim to: (1) extend the MethCORR method to 10 additional cancer types and (2) to illustrate that the inferred gene expression is accurate and clinically informative.RESULTS: Regression models to infer gene expression information from DNA methylation were developed for ten common cancer types using matched RNA sequencing and DNA methylation profiles (HumanMethylation450 BeadChip) from The Cancer Genome Atlas Project. Robust and accurate gene expression profiles were inferred for all cancer types: on average, the expression of 11,000 genes was modeled with good accuracy and an intra-sample correlation of R2 = 0.90 between inferred and measured gene expression was observed. Molecular pathway analysis and transcriptional subtyping were performed for breast, prostate, and lung cancer samples to illustrate the general usability of the inferred gene expression profiles: overall, a high correlation of r = 0.96 (Pearson) in pathway enrichment scores and a 76% correspondence in molecular subtype calls were observed when using measured and inferred gene expression as input. Finally, inferred expression from FFPE tissue correlated better with RNA sequencing data from matched fresh-frozen tissue than did RNA sequencing data from FFPE tissue (P < 0.0001; Wilcoxon rank-sum test).
    CONCLUSIONS: In all cancers investigated, MethCORR enabled DNA methylation-based transcriptional analysis, thus enabling future analysis of cancer in situations where high-quality DNA, but not RNA, is available. Here, we provide the framework and resources for MethCORR modeling of ten common cancer types, thereby widely expanding the possibilities for transcriptional studies of archival FFPE material.
    Keywords:  Biomarkers; Cancer; DNA methylation; FFPE tissue; Gene expression; Molecular subtypes; RNA sequencing
  67. Lancet. 2021 Jan 23. pii: S0140-6736(21)00093-3. [Epub ahead of print]397(10271): 279-280
    Finucane TE, McKenzie R.
  68. Nat Cell Biol. 2021 Jan 25.
    Recasens-Alvarez C, Alexandre C, Kirkpatrick J, Nojima H, Huels DJ, Snijders AP, Vincent JP.
      Ribosomes are multicomponent molecular machines that synthesize all of the proteins of living cells. Most of the genes that encode the protein components of ribosomes are therefore essential. A reduction in gene dosage is often viable albeit deleterious and is associated with human syndromes, which are collectively known as ribosomopathies1-3. The cell biological basis of these pathologies has remained unclear. Here, we model human ribosomopathies in Drosophila and find widespread apoptosis and cellular stress in the resulting animals. This is not caused by insufficient protein synthesis, as reasonably expected. Instead, ribosomal protein deficiency elicits proteotoxic stress, which we suggest is caused by the accumulation of misfolded proteins that overwhelm the protein degradation machinery. We find that dampening the integrated stress response4 or autophagy increases the harm inflicted by ribosomal protein deficiency, suggesting that these activities could be cytoprotective. Inhibition of TOR activity-which decreases ribosomal protein production, slows down protein synthesis and stimulates autophagy5-reduces proteotoxic stress in our ribosomopathy model. Interventions that stimulate autophagy, combined with means of boosting protein quality control, could form the basis of a therapeutic strategy for this class of diseases.